Effects of 5-Aza-dC on 5-Fu chemosensitivity by modulating TIP30 gene expression in human colorectal cancer cells

Abstract

To investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC) on TIP30 gene expression and the relationship between TIP30 expression and the sensitivity to 5-fluouracil (5-Fu) in colorectal cancer cells. The methylation profile of TIP30 gene in HCT116 colorectal cancer cells was determined by methylation-specific PCR. The levels of TIP30 mRNA and protein were determined by RT-PCR and Western blot after the 5-Aza-dC treatment. MTT assay was used to detect the chemosensitivity of HCT116 cells to 5-Fu. TIP30 gene displayed complete DNA methylation in the HCT116 cells without 5-Aza-dC pretreatment. After the 5-Aza-dC treatment for 3 days, only demethylating PCR amplification product was detected and TIP30 gene showed DNA demethylation. With the prolongation of the time of removal of 5-Aza-dC treatment, methylated and demethylated PCR amplification products were observed and TIP30 gene displayed both DNA methylation and DNA demethylation in the colorectal cancer cells. At the day 10 after removal of 5-Aza-dC, methylating PCR amplification product appeared and TIP30 gene showed DNA methylation. No expressions of TIP30 mRNA and protein were detected in the HCT116 cells untreated with 5-Aza-dC. After the treatment of 5-Aza-dC for 3 d and then removed the 5-Aza-dC, the expressions of TIP30 mRNA and protein were increased obviously. With the prolonged time after 5-Aza-dC removal, the expressions of TIP30 mRNA and protein decreased and reached the lowest level on day 10. The IC50 values of 5-Fu were 41.62, 33.17 and 4.96 µg/ml in the HCT116 cells pretreated with 5-Aza-dC, d0 and d10 with the drug removal after drug treatment for 3 d, respectively. The results of this study show that the expression of TIP30 gene may be associated with its DNA methylation status and may affect the sensitivity of colorectal cancer cells to 5-Fu.